RESUMO
OBJECTIVE: Since onsite education is difficult due to the COVID-19 pandemic, official development assistance (ODA) projects have implemented online training of trainers (ToT) for emergency medical experts and staff. This study aims to share and discuss the ToT experience and its results in Uzbekistan. METHODS: We trained emergency medical advanced course instructors through online ToT among emergency medical service experts in Uzbekistan as a part of an ODA project. After the ToT, instructors were selected based on written tests, video monitoring of practice, and simulation performance. They operated the emergency medical course including lectures, practices, and simulations for 5 days. We tested the trainees through written tests before and after the course. They were surveyed regarding the course contents, its relevance, and their satisfaction with the course. RESULTS: Six instructors were selected after the online ToT program. They educated 68 emergency medical workers through the three training courses. The total score of the pretest was 129.2±34.8, and the posttest score was 170.8±31.2, which was significantly higher (P<0.05). The satisfaction calculated by adding the values of survey items for this curriculum was 28.0 (interquartile range, 26.0-30.0), and there was no statistical difference regarding trainee satisfaction between the three courses (P=0.148). CONCLUSION: Instructors trained by online ToT programs could provide an in-person emergency medical advanced course.
RESUMO
Bag-valve-mask is a device that manually provides positive oxygen pressure. The grip technique of the character E and C shape is recommended to carry out this effectively. However, when applying this method, the hand in which the direction of the EC technique should be performed and the degree of adhesion pressure while performing the technique are unknown. This study aims to identify the factors influencing tidal volume and to determine the ideal sealing method between mask and face in the one-handed EC technique to optimize the Vt. A simulation study was conducted using a mechanical lung model in a scenario that resembled respiratory arrest. Multiple regression analyses identified high peak pressure, high left spot adhesion strength of the mask, and low right spot and bottom spot adhesion strength of the mask as significant factors. To provide an optimal Vt, it may be necessary to apply more strength to the left area of the mask when forming the "C" shape with the thumb and index finger of left hand using the one-handed EC technique.
RESUMO
BACKGROUND: RNA interference (RNAi) is a powerful technique to effectively silence or knock down gene function in mammalian cells. For better cell-type RNAi experiments in vivo, AAV vector-based RNA interference systems need to be improved. New method: In this study, we developed an AAV vector (CREon shRNA) that expressed CRE-dependent short hairpin RNA (shRNA) and fluorescent proteins simultaneously. RESULTS: We verified the Cre-dependent knockdown efficiency of the newly developed CREon shRNA vector in both HEK293T cells overexpressing TREK-1 and PC3 cells with endogenous TREK-1. Next, we packaged this TREK-1 CREon vector with AAV and injected it into the hippocampus of the brain together with a synapsin or GFAP promoter-driven CRE virus, confirming that it works well cell-selectively even in vivo. Finally, this viral vector was applied to an animal model of LPS-induced depression to determine whether behavioral changes occurred. Comparison with existing methods: With the existing pSico or pAAV-Sico-Red vectors, expression of fluorescent protein disappears when shRNA is conditionally activated by CRE recombinase, but our Creon shRNA vector showed simultaneous expression of both shRNA and fluorescent protein. Thus, it offers the advantage of allowing easy visual distinction of knocked-down cells. CONCLUSION: The newly improved CREon shRNA vector can be used as a novel research tool for conditional shRNA, and may be useful for various in vivo studies such as cancer and neurobiology.
Assuntos
Vetores Genéticos , Mamíferos , Animais , Células HEK293 , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: For adequate ventilation during bag-valve-mask ventilation, rescuers should ensure a proper mask seal using the one-handed or two-handed technique. Little is known about the magnitude of sealing forces of a bag-valve-mask needed for adequate ventilation. This study aimed to explore the effect of the 4-point sealing forces of a bag-valve-mask on tidal volume while using the one-handed technique, focusing on the moderating effect of C length (the distance from the thumb to the index finger in the C shape of the one-hand EC grip). METHODS: A prospective, descriptive simulation study was conducted. A convenience sample of 125 undergraduate paramedic students from two universities was recruited. A self-reported questionnaire was used to collect subjective variables. Tidal volumes, 4-point sealing forces of the mask, peak pressure, and C length of the C shape in the one-hand EC grip were measured using the mechanical lung model under a simulated adult respiratory arrest. Hierarchical regression analysis was used to determine the moderating effect of C length on tidal volume in bag-valve-mask ventilation. RESULTS: The average C length, peak pressure, and tidal volume were 7.54 (± 1.85) cm, 11.62 (± 5.40) cmH2O, and 321.66 (± 135.18) mL, respectively. The average range of the 4-point sealing forces was 0.03-0.69 N. The apex sealing force was the weakest among the 4-point sealing forces. Hierarchical regression analysis demonstrated that tidal volume accounted for 62.7% of the variance by C length, peak pressure, and apex sealing force during bag-valve-mask ventilation (F = 9.676, p < 0.001). C length moderated the effect of the apex sealing force and peak pressure on the tidal volume, meaning the higher the peak pressure and apex sealing force, the more the tidal volume and the longer the C length. CONCLUSION: This first simulation study measuring the 4-point sealing forces during bag-valve-mask ventilation provides effective advice that can be adopted in clinical practice without side effects and underpins the importance of continuous retraining and assessment focused on individual physical characteristics, such as C length and bag-valve-mask sealing forces.
Assuntos
Respiração Artificial , Insuficiência Respiratória , Volume de Ventilação Pulmonar , Adulto , Humanos , Máscaras , Estudos Prospectivos , Insuficiência Respiratória/terapiaRESUMO
Hevin, also known as SPARC-like protein 1 (SPARCL1 or SC1), is a synaptogenic protein secreted by astrocytes and modulates the formation of glutamatergic synapses in the developing brain by interacting with synaptic adhesion proteins, such as neurexin and neuroligin. Here, we identified the neuron-specific vesicular protein calcyon as a novel interaction partner of hevin and demonstrated that this interaction played a pivotal role in synaptic reorganization after an injury in the mature brain. Astrocytic hevin was upregulated post-injury in a photothrombotic stroke model. Hevin was fragmented by MMP3 induced during the acute stage of brain injury, and this process was associated with severe gliosis. At the late stage, the functional hevin level was restored as MMP3 expression decreased. The C-terminus of hevin interacted with the N-terminus of calcyon. By using RNAi and binding competitor peptides in an ischemic brain injury model, we showed that this interaction was crucial in synaptic and functional recoveries in the sensory-motor cortex, based on histological and electrophysiological analyses. Regulated expression of hevin and calcyon and interaction between them were confirmed in a mouse model of traumatic brain injury and patients with chronic traumatic encephalopathy. Our study provides direct evidence for the causal relationship between the hevin-calcyon interaction and synaptic reorganization after brain injury. This neuron-glia interaction can be exploited to modulate synaptic reorganization under various neurological conditions.
Assuntos
Lesões Encefálicas/terapia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Membrana/uso terapêutico , Animais , Humanos , Masculino , Camundongos , Sinapses/metabolismoRESUMO
AEG-1, also called MTDH, has oncogenic potential in numerous cancers and is considered a multifunctional modulator because of its involvement in developmental processes and inflammatory and degenerative brain diseases. However, the role of AEG-1 in astrocytes remains unknown. This study aimed to investigate proteins directly regulated by AEG-1 by analyzing their RNA expression patterns in astrocytes transfected with scramble shRNA and AEG-1 shRNA. AEG-1 knockdown down-regulated TWIK-1 mRNA. Real-time quantitative PCR (qPCR) and immunocytochemistry assays confirmed that AEG-1 modulates TWIK-1 mRNA and protein expression. Electrophysiological experiments further revealed that AEG-1 further regulates TWIK-1-mediated potassium currents in normal astrocytes. An RNA immunoprecipitation assay to determine how AEG-1 regulates the expression of TWIK-1 revealed that AEG-1 binds directly to TWIK-1 mRNA. Furthermore, TWIK-1 mRNA stability was significantly increased upon overexpression of AEG-1 in cultured astrocytes (p < 0.01). Our findings show that AEG-1 serves as an RNA-binding protein to regulate TWIK-1 expression in normal astrocytes.
RESUMO
Astrocytes, the most abundant cell type in the brain, are non-excitable cells and play critical roles in brain function. Mature astrocytes typically exhibit a linear current-voltage relationship termed passive conductance, which is believed to enable astrocytes to maintain potassium homeostasis in the brain. We previously demonstrated that TWIK-1/TREK-1 heterodimeric channels mainly contribute to astrocytic passive conductance. However, the molecular identity of astrocytic passive conductance is still controversial and needs to be elucidated. Here, we report that spadin, an inhibitor of TREK-1, can dramatically reduce astrocytic passive conductance in brain slices. A series of gene silencing experiments demonstrated that spadin-sensitive currents are mediated by TWIK-1/TREK-1 heterodimeric channels in cultured astrocytes and hippocampal astrocytes from brain slices. Our study clearly showed that TWIK-1/TREK-1-heterodimeric channels can act as the main molecular machinery of astrocytic passive conductance, and suggested that spadin can be used as a specific inhibitor to control astrocytic passive conductance.
Assuntos
Astrócitos/fisiologia , Encéfalo/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Multimerização Proteica , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
TREK-1 (TWIK-related K+ channel), a member of the two-pore domain K+ (K2P) channel family, is highly expressed in astrocytes, where it plays a key role in glutamate release and passive conductance. In addition, TREK-1 is induced to protect neurons under pathological conditions such as hypoxia. However, the upstream regulation of TREK-1 remains poorly understood. In this study, we found that AEG-1 (astrocyte elevated gene-1) regulates the expression of astrocytic TREK-1 under hypoxic conditions. Upregulation of AEG-1 increased expression of TREK-1 in astrocytes, and knockdown of AEG-1 dramatically decreased the mRNA and protein levels of TREK-1, which were restored by expression of shRNA-insensitive AEG-1. In addition, expression of TREK-1 was not regulated in the absence of AEG-1, even when HIF1α was present. Together, these results suggest that AEG-1 acts as a major upstream regulator of TREK-1 channels in astrocytes under hypoxia. SIGNIFICANCE OF THE STUDY: Previous studies have reported that hypoxia increases the expression of astrocytic TREK-1 and that increased TREK-1 expression protects neuronal cells from apoptosis. However, its cellular mechanism is not clear. In this study we first showed that AEG-1 is a major mediator of hypoxic-regulated TREK-1 expression in normal astrocytes independently of HIF-1α.
Assuntos
Astrócitos/metabolismo , Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose , Astrócitos/citologia , Eletroporação , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neurônios/metabolismo , RNA Mensageiro/metabolismoRESUMO
TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.
Assuntos
Habenula , Animais , Ansiedade/genética , Neurônios Colinérgicos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
TWIK-related potassium channel-1 (TREK-1) is broadly expressed in the brain and involved in diverse brain diseases, such as seizures, ischemia, and depression. However, the cell type-specific roles of TREK-1 in the brain are largely unknown. Here, we generated a Cre-dependent TREK-1 knockdown (Cd-TREK-1 KD) transgenic mouse containing a gene cassette for Cre-dependent TREK-1 short hairpin ribonucleic acid to regulate the cell type-specific TREK-1 expression. We confirmed the knockdown of TREK-1 by injecting adeno-associated virus (AAV) expressing Cre into the hippocampus of the mice. To study the role of hippocampal neuronal TREK-1 in a lipopolysaccharide (LPS)-induced depression model, we injected AAV-hSyn-BFP (nCTL group) or AAV-hSyn-BFP-Cre (nCre group) virus into the hippocampus of Cd-TREK-1 KD mice. Interestingly, the immobility in the tail suspension test after LPS treatment did not change in the nCre group. Additionally, some neurotrophic factors (BDNF, VEGF, and IGF-1) significantly increased more in the nCre group compared to the nCTL group after LPS treatment, but there was no difference in the expression of their receptors. Therefore, our data suggest that TREK-1 in the hippocampal neurons has antidepressant effects, and that Cd-TREK-1 KD mice are a valuable tool to reveal the cell type-specific roles of TREK-1 in the brain.
Assuntos
Transtorno Depressivo/etiologia , Hipocampo/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Animais , Comportamento Animal/efeitos dos fármacos , Corticosterona/sangue , Citocinas/genética , Citocinas/metabolismo , Giro Denteado/metabolismo , Dependovirus/genética , Transtorno Depressivo/metabolismo , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Transgênicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1-deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1-deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1-deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl- secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis.
Assuntos
Anoctamina-1/fisiologia , Carbacol/farmacologia , Cloretos/metabolismo , Toxina da Cólera/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Animais , Anoctamina-1/genética , Cálcio/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/fisiologia , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite/patologia , Feminino , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Via Secretória/efeitos dos fármacos , Via Secretória/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat-containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression profiling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Técnicas de Patch-Clamp , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, ß-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTYH2-C) as a bait. Using in vitro and in vivo binding assays, we confirmed the protein-protein interactions between TTYH2 and ß-COP. We also found that the surface expression and activity of TTYH2 were decreased by co-expression with ß-COP in the heterologous expression system. In addition, ß-COP associated with TTYH2 in a native condition at a human colon cancer cell line, LoVo cells. The over-expression of ß-COP in the LoVo cells led to a dramatic decrease in the surface expression and activity of endogenous TTYH2. Collectively, these data suggested that ß-COP plays a critical role in the trafficking of the TTYH2 channel to the plasma membrane. [BMB Reports 2019; 52(7): 445-450].
Assuntos
Proteína Coatomer/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Propriedades de SuperfícieRESUMO
Two-pore domain K+ (K2P) channels have been shown to modulate neuronal excitability. The physiological role of TWIK-1, the first identified K2P channel, in neuronal cells is largely unknown, and we reported previously that TWIK-1 contributes to the intrinsic excitability of dentate gyrus granule cells (DGGCs) in mice. In the present study, we investigated the coexpression of TWIK-1 and TASK-3, another K2P member, in DGGCs. Immunohistochemical staining data showed that TASK-3 proteins were highly localized in the proximal dendrites and soma of DGGCs, and this localization is similar to the expression pattern of TWIK-1. TWIK-1 was shown to associate with TASK-3 in DGGCs of mouse hippocampus and when both genes were overexpressed in COS-7 cells. shRNA-mediated gene silencing demonstrated that TWIK-1/TASK-3 heterodimeric channels displayed outwardly rectifying currents and contributed to the intrinsic excitability of DGGCs. Neurotensin-neurotensin receptor 1 (NT-NTSR1) signaling triggered the depolarization of DGGCs by inhibiting TWIK-1/TASK-3 heterodimeric channels, causing facilitated excitation of DGGCs. Taken together, our study clearly showed that TWIK-1/TASK-3 heterodimeric channels contribute to the intrinsic excitability of DGGCs and that their activities are regulated by NT-NTSR1 signaling.
Assuntos
Giro Denteado/metabolismo , Potenciais Pós-Sinápticos Excitadores , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Multimerização Proteica , Animais , Células COS , Chlorocebus aethiops , Giro Denteado/citologia , Giro Denteado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Neurotensina/metabolismoRESUMO
OBJECTIVE: The injury-related disease burden in South Korea is relatively high compared to that in other nations. This study was conducted to identify the scale and causes of injury by severity, using an injury pyramid and analyzing the sex and age-dependent differences and the basic demographic characteristics. METHODS: Unintentional injury was estimated for each group after classifying injury-related deaths, hospitalizations, and outpatient department (OPD) visits based on their severity. The injury pyramid was calculated by classifying the injury outcomes into deaths, hospitalizations, and outpatient visits. RESULTS: The incidence of unintentional injury included 31.74 deaths, 1,715.27 hospitalizations, and 7,317.55 OPD visits per 100,000 persons. The incidence of injury was higher among males than that among females. There were more hospitalizations and OPD visits than deaths among females. The mortality and hospitalization rates due to injury per 100,000 persons were the highest among those ≥75 years old, and the OPD visit rate was the highest among preschool children aged 0 to 6 years. The injury mechanisms that caused most deaths and hospitalizations in South Korea were injuries related to traffic, falls, poisoning, and burns/fires. CONCLUSION: The scale of unintentional injury varies based on sex, age, and injury mechanism; additionally there are differences in the incidence of injuries between males and females depending on their age and injury mechanism. The high incidence of injury in elderly people could be a factor that increases the burden of injury in South Korea; hence, it is necessary to develop an injury prevention program that targets the elderly.
RESUMO
Copine1 (CPNE1), known as a calcium-dependent membrane-binding protein, has tandem C2 domains and an A domain. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Protein kinase B (AKT) phosphorylation in the hippocampal progenitor cell line, HiB5. To better understand its cellular function, we carried out a yeast two-hybrid screening to find CPNE1 binding partners. Among the identified proteins, 14-3-3γ appears to directly interact with CPNE1. Between CPNE1 and 14-3-3γ, the physical interaction as well as the specific binding regions of CPNE1 was confirmed in vitro and in vivo. Furthermore, among the seven 14-3-3 isotypes, only 14-3-3γ directly interacts with CPNE1. Our results also demonstrate that AKT phosphorylation, neurite outgrowth and expression of the neuronal marker protein are increased when 14-3-3γ is overexpressed in CPNE1 high expressed HiB5 cells. Furthermore, the neighboring Ser54 amino acids residue of C2A domain in CPNE1 has an important role in binding with 14-3-3γ, and in differentiation-related function of CPNE1. Moreover, mutation of Ser54 amino acids residue in CPNE1 effectively decreased association with 14-3-3γ and neuronal differentiation of HiB5 cells. Collectively, our findings indicate that 14-3-3γ regulates the differentiation ability of CPNE1 through the binding with C2A domain of CPNE1 in HiB5 cells.